Association of the uterine microbiome to mare fertility

Abstract

Currently, limited information on the microbiome (community of microorganisms) and its role in infertility in the equine uterus is available beyond the detection of specific pathogens in some subfertile mares. Given that less than 1% of environmental bacteria are cultivable on common media (Handelsman, J. 2004, Microbiol Mol Biol Rev 68, 669-685.), it is likely that these cultured species represent a minority of the resident population. The overarching objectives of this project were to i) identify the resident uterine microbiome in estrus ii) assess how this microbiome evolves over time and iii) compare traditional culture-dependent methods of assessing the presence of bacteria in the equine uterus with 16S rRNA sequencing. Uterine cytobrush and swab samples were collected from 157 maiden Thoroughbred mares aged 8 years or younger and 116 Thoroughbred barren mares aged 4 to 15 years at the first reproductive exam of the breeding season. A second sample was collected from mares that were not pregnant at the end of the season (n=23). Furthermore, 20 of the mares were sampled in three consecutive breeding seasons. All cytobrush samples were placed and stored in RNAlater and kept at -20° C until further processing. Uterine swabs were plated on sheep blood and MacConkey plates, incubated at 37° C in aerobic conditions and assessed for bacterial growth at 24, 48 and 72 hours. DNA was extracted from cytobrush samples using routine methods. The genomic DNArecovered was used as a template for PCR amplification of the V3–V4 variable region of the bacterial 16S rRNA gene. Amplicons were sequenced on the Illumina MiSeq platform. DNA sequencing data was then processed to assess the equine microbiome composition. The richness (number of bacterial species) and evenness (distribution of bacterial numbers) of bacterial genetic sequences was compared between maiden, barren and wet mares and correlated with reproductive success. The microbiome was highly dynamic and changed significantly between seasons within mares with a limited “core microbiome” being detected. In some mares, conventional culture techniques failed to identify known equine genital pathogens that were detected using 16S rRNA sequencing. While the richness and evenness of the microbiome was comparable in maiden, dry and wet mares, the overall bacterial abundance was higher in mares that were barren at the beginning of the season and failed to become pregnant during the season, when compared to successfully bred barren mares and all maiden mares. This was the first time the microbiome of the equine uterus was studied over several timepoints/seasons. Future studies could explore a prognostic test that allows prediction of fertility in barren mares. This work was funded by AgriFutures Australia.